title: Arabidopsis thaliana AMY3 is a unique redox-regulated chloroplastic α-amylase.
abstract: α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for β-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp(666)), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a β-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and β-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5-8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys(499) and Cys(587) is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.
authors.affiliation: Institute of Agricultural Sciences, ETH Zürich, Universitätstrasse 2, 8092 Zürich, Switzerland.
authors.forename: David
authors.initials: D
authors.lastname: Seung
authors.affiliation: Institute of Agricultural Sciences, ETH Zürich, Universitätstrasse 2, 8092 Zürich, Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Thalmann
authors.affiliation: Department of Experimental Evolutionary Biology, University of Bologna, I-40126 Bologna, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Sparla
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Maher
authors.initials: M
authors.lastname: Abou Hachem
authors.affiliation: Institute of Agricultural Sciences, ETH Zürich, Universitätstrasse 2, 8092 Zürich, Switzerland.
authors.forename: Sang Kyu
authors.initials: SK
authors.lastname: Lee
authors.affiliation: Institut de Biologie des Plantes, UMR 8618 CNRS/University Paris-Sud, 91405 Orsay, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Issakidis-Bourguet
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
authors.affiliation: Institute of Agricultural Sciences, ETH Zürich, Universitätstrasse 2, 8092 Zürich, Switzerland.
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Zeeman
authors.affiliation: Institute of Agricultural Sciences, ETH Zürich, Universitätstrasse 2, 8092 Zürich, Switzerland. Electronic address: dsantelia@botinst.uzh.ch.
authors.forename: Diana
authors.initials: D
authors.lastname: Santelia
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D060365:Chloroplast Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics; D001467:Hordeum / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D013213:Starch / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D060365:Chloroplast Proteins; D013213:Starch; D000516:alpha-Amylases
keywords: Amylopectin; Arabidopsis; Carbohydrate Metabolism; Plant Biochemistry; Redox Regulation; Starch; Thioredoxin; α-Amylase